Seed bioinformatics

Analysis of gene expression data sets is a potent tool for gene function prediction, cis-element discovery, and hypothesis generation for the model plant Arabidopsis thaliana, and more recently for other agriculturally relevant species. In the case of Arabidopsis thaliana, experiments conducted by individual researchers to document its transcriptome have led to large numbers of data sets being made publicly available for data mining by the so-called “electronic northerns,” co-expression analysis and other methods. Given that approximately 50% of the genes in Arabidopsis have no function ascribed to them by “conventional” homology searches, and that only around 10% of the genes have had their function experimentally determined in the laboratory, these analyses can accelerate the identification of potential gene function at the click of a mouse. This chapter covers the use of bioinformatic data mining tools available at the Bio-Array Resource (http://www.bar.utoronto.ca) and elsewhere for hypothesis generation in the context of seed biology

Germination-still a mystery

Germination is a complex process during which the seed must quickly recover physically from maturation drying, resume a sustained intensity of metabolism, complete essential cellular events to allow for the embryo to emerge, and prepare for subsequent seedling growth. Early following the start of imbibition of the dry seed there is re-establishment of metabolism; restitution of the chemical and structural integrity of cells requires the co-participation of synthetic and protective events. Protein synthesis and respiratory activity initially involve components stored within the mature dry seed, although transcription and translation commence early during imbibition, as shown by transciptome and metabolome analyses. Increases or modifications to hormones, especially GA, play an important role in achieving the completion of germination, at least in intact seeds. Removal or deactivation of ABA is also important; interactions between this and GA play a regulatory role. A restraint on the completion of germination in seeds of some species is imposed by the surrounding structures, e.g. the endosperm, and thus there is a requirement either for it to be enzymically weakened to allow the radicle to emerge, or for sufficient force to be generated within the embryo axis to physically break through, or both. While there is much information with respect to changes in gene expression during germination, no key event(s) has been identified that results in its completion. The downstream effects of the observed hormone changes are not known, and given the multipart nature of the seed, the requirements imposed upon it (repair, maintenance, preparation for seedling growth) in addition to completing germination (which involves only a limited number of cells), the challenge to identify ‘germination-completion’ genes is large. Hence there are limited opportunities at present for improving germination through genetic manipulation

Germination of Arabidopsis thaliana seeds is not completed as a result of elongation of the radicle but of the adjacent transition zone and lower hypocotyl

The completion of germination of seeds of Arabidopsis thaliana is marked by the appearance of the radicle through the surrounding endosperm and testa. Using confocal microscopy and green fluorescent protein (GFP)-transformed embryos to highlight the epidermal cell walls it has been possible to conduct time-lapse photography of individual embryos during their germination. This reveals that the elongation of embryo cells to effect completion of germination does not occur within the radicle itself, but rather within a discrete region that is immediately proximal to the radicle. This region, identifiable as the lower hypocotyl and hypocotyl–radicle transition zone, is also definable by accumulation of carbohydrate-containing bodies during germination, and distinct GFP expression of GAL4–GFP in enhancer trap lines. Flow cytometric studies show that there is an increase in the proportion of 4C nuclei in the axis which coincides with a considerable increase in length of the hypocotyl, and the occurrence of endopolyploid (8C and 16C) nuclei accompanies the 2-fold increase in mean cell size in the region of elongation, the lower hypocotyl, and hypocotyl–radicle transition zone. Thus the observed cell elongation during germination is accompanied by an increase in nuclear DNA content, and the resultant elongation of the axis to effect radicle emergence is due to cell expansion, not to cell division. When studying the molecular events involved in the completion of germination, therefore, it may be prudent to focus on this region of elongation

α-Galactosidase is synthesized in tomato seeds during development and is localized in the protein storage vacuoles

The localization of the enzyme α-galactosidase (EC 3.2.1.22) was investigated during its synthesis in developing tomato (Lycopersicon esculentum Mill.) cv. Trust seeds. This enzyme is also present in germinating seeds, where it is involved in the mobilization of carbohydrate reserves during and following seed germination. Subcellular fractionation of developing tomato seeds revealed that there is a cosedimentation between α-galactosidase activity and protein storage vacuoles in a density gradient, which is dependent upon the presence of membranes. A second approach to localizing this enzyme involved the transient transformation of protoplasts from developing tomato seeds. A reporter construct, coding for tomato α-galactosidase, fused N-terminally to the bacterial enzyme chloramphenicol acetyltransferase was used for transient expression. Immunofluorescence microscopy revealed a colocalization between the α-galactosidase - chloramphenicol acetyltransferase fusion protein and the α-tonoplast intrinsic protein, and a partial colocalization with the dark intrinsic protein (both vacuolar proteins). These data indicate that the protein storage vacuole is the intracellular location for α-galactosidase in developing tomato seeds.Key words: α-galactosidase, protein storage vacuole, seed development, seed protoplasts, tomato, tonoplast intrinsic protein

The emergence of embryos from hard seeds is related to the structure of the cell walls of the micropylar endosperm, and not to endo-β-mannanase activity

• Background and Aims Seeds of carob, Chinese senna, date and fenugreek are hard due to thickened endosperm cell walls containing mannan polymers. How the radicle is able penetrate these thickened walls to complete seed germination is not clearly understood. The objective of this study was to determine if radicle emergence is related to the production of endo-β-mannanase to weaken the mannan-rich cell walls of the surrounding endosperm region, and/or if the endosperm structure itself is such that it is weaker in the region through which the radicle must penetrate. • Methods Activity of endo-β-mannanase in the endosperm and embryo was measured using a gel assay during and following germination, and the structure of the endosperm in juxtaposition to the radicle, and surrounding the cotyledons was determined using fixation, sectioning and light microscopy. • Key Results The activity of endo-β-mannanase, the major enzyme responsible for galactomannan cell wall weakening increased in activity only after emergence of the radicle from the seed. Thickened cell walls were present in the lateral endosperm in the hard-seeded species studied, but there was little to no thickening in the micropylar endosperm except in date seeds. In this species, a ring of thin cells was visible in the micropylar endosperm and surrounding an operculum which was pushed open by the expanding radicle to complete germination. • Conclusions The micropylar endosperm presents a lower physical constraint to the completion of germination than the lateral endosperm, and hence its structure is predisposed to permit radicle protrusion

Genetic control of plant development by overriding a geometric division rule

Formative cell divisions are critical for multicellular patterning. In the early plant embryo, such divisions follow from orienting the division plane. A major unanswered question is how division plane orientation is genetically controlled, and in particular whether this relates to cell geometry. We have generated a complete 4D map of early Arabidopsis embryogenesis and used computational analysis to demonstrate that several divisions follow a rule that uses the smallest wall area going through the center of the cell. In other cases, however, cell division clearly deviates from this rule, which invariably leads to asymmetric cell division. By analyzing mutant embryos and through targeted genetic perturbation, we show that response to the hormone auxin triggers a deviation from the “shortest wall” rule. Our work demonstrates that a simple default rule couples division orientation to cell geometry in the embryo and that genetic regulation can create patterns by overriding the default rul